The present invention relates to a method for determination of nucleated red blood cells in a blood sample. More specifically the method differentiates nucleated red blood cells from other cell types using a direct current impedance measurement in a non-focused flow aperture, and enumerates nucleated red blood cells in a blood sample.
Normal peripheral blood contains mature red blood cells which are free of nucleus. Nucleated red blood cells (NRBCs), or erythroblasts, are immature red blood cells. They normally occur in the bone marrow but not in peripheral blood. However, in certain diseases such as anemia and leukemia, NRBCs also occur in peripheral blood. Therefore, it is of clinical importance to measure NRBCs. Traditionally, differentiation and enumeration of NRBC are performed manually. The process involves the smearing of a blood sample on a microscope slide and staining the slide, followed by manual visual analysis of the individual slide. The NRBC concentration is reported as numbers of NRBC per 100 white blood cells. Usually, 200 white blood cells and the numbers of NRBC present in the same region on a blood smear are counted and the numbers are divided by 2 to express the NRBC concentration as the numbers of NRBC/100 WBC. This approach is extremely time-consuming as well as being subjective to the interpretation of the individual analyzing the slide.
In recent years, several fluorescence flow cytometry methods have been developed for differentiating NRBCs. These methods utilizes specific nuclear staining technique to distinguish NRBCs from other cell types because it is difficult to differentiate NRBCs based on their electronic or optical properties.
U.S. Pat. No. 5,298,426 (to Inami et al.) discloses a fluorescence method for differentiating NRBCs. The method utilizes a two-step staining using a first fluid and a second fluid. Inami et al. teaches that the first fluid contains an erythroblast-staining dye that diffuses into nucleated red blood cells to specifically stain their nuclei, and then separating a group of NRBCs from other cell groups on a two-dimensional plot whereby the results of NRBC differentiation are computed.
U.S. Pat. No. 5,559,037 (to Kim et al.) discloses a method for flow cytometric analysis of NRBCs and leukocytes. The method comprises lysis of red blood cells and NRBC cytoplasm from a whole blood sample to expose the NRBC nuclei to a vital nuclear stain and minimizing the permeation of the vital nuclear stain into the leukocytes and analyzing the sample by measuring fluorescence and two angles of light scatter. This method features a triple triggering method which blocks the signals from debris (fluorescent and non-fluorescent) and identifies the signals which fall below the ALL trigger but above the fluorescence trigger (FL3) as NRBCs. ALL is the axial loss of light or the light scatter signals detected at 0xc2x0 from the incident light. Therefore, pre-bating signals in more than one dimension are required in this method for identification of NRBC population. In addition, the method requires heating of the reagent to 42xc2x0 C. in order to obtain the NRBC and leukocyte differentiations.
U.S. Pat. No. 5,648,225 (to Kim et al) discloses a method of using a multipurpose lysing reagent for subclassification of nucleated blood cells. The method comprises the steps of lysing a blood sample with the multipurpose lysing reagent which contains a nuclear stain, incubating the sample mixture at an elevated temperature, and determining the nucleated blood cells including NRBCs with an automated electro-optical hematology instrumentation.
U.S. Pat. No. 5,879,900 (to Kim et al) discloses a method of differentiating NRBCs, damaged white blood cells (WBC), WBC and a WBC differential in a blood sample by flow cytometry. The method includes lysing a blood sample; staining NRBCs and any damaged white blood cells with a vital nuclear stain; analyzing the sample mixture by measuring at least one fluorescence, and at least one light scatter signals in a range from 0xc2x0 to 1xc2x00 and 3xc2x0 to 10xc2x0; constructing a three-dimensional plot from the fluorescence and light scatter signals; and differentiating and enumerating WBC, NRBC, damaged WBC and a WBC subclass differential.
EP 1 004 880 A2 discloses reagents and a method for discrimination and counting of nucleated red blood cells. The method includes the steps of lysing red blood cells, staining white blood cells and NRBCs, assaying the sample by measuring at least one scattered light parameter, and at least one fluorescence parameter.
U.S. Pat. No. 5,874,310 (to Li et al) discloses a method for differentiation of nucleated red blood cells. The method includes lysing mature red blood cells and analyzing the sample in a flow cell by light scatter measurement to differentiate NRBCs from other cell types. The light scatter measurement is performed by using two low angle light scatter signals of less than 10xc2x0. The method further includes a concurrent differentiation of white blood cells using electronic and optical analysis, wherein the electronic analysis is a DC impedance measurement.
U.S. Pat. No. 5,917,584 (to Li et al) discloses a method for differentiation of nucleated red blood cells. The method includes lysing mature red blood cells in a blood sample; analyzing the sample in a flow cell by two angles of light scatter measurement to differentiate NRBCs from other cell types, wherein the second light scatter signal is a medium angle or a right-angle light scatter signal.
The above described methods enable differentiation and enumeration of NRBCs and leukocytes by fluorescence flow cytometry and light scatter measurements. However, fluorescence and light scatter measurements are complex and expensive detection methods.
Many current non-fluorescence automated hematology analyzers, such as Abbott Cell-Dyn(copyright) 3500, COULTER(copyright) Gen*S(trademark), Bayer Advia*120(copyright), and Sysmex(trademark) NE-9000 are only able to provide NRBC flagging for the possible presence of NRBCs in an analyzed blood sample when the instruments sense an increased amount of signals near blood cell debris area of an obtained cell distribution histogram. However, such techniques frequently generate false positive flagging because many other blood abnormalities can cause increased signals at the same area, such as platelet clumps and sickle cells, as well as red cell debris from insufficiently lysed blood samples. In these methods NRBCs are not distinctly identified. Instead, only a common NRBC sample distribution pattern in a histogram or a dotplot is recognized by the instrument which can be confused with a similar pattern generated by above-mentioned other causes.
Furthermore, a well known problem with NRBC containing samples is erroneous white blood cell count (WBC) reported by hematology analyzers on these samples. Since the nuclear volumes of NRBC are close to those of white blood cells, and they are commonly counted as white blood cells on hematology analyzers which measure the sizes of blood cells, resulting an elevation of WBC. Therefore, correction of NRBC contribution to the WBC reported from hematology analyzer is required for samples containing NRBC. Current practice in the clinical laboratory is to subtract the numbers of NRBC obtained by manual count from the WBC count reported by the hematology analyzers. This is time consuming and error prone.
On the other hand, measurement of hemoglobin (Hgb) concentration of blood samples is an integral part of blood analysis, which is important for disease diagnosis and for monitoring responses to medical treatment. It is desirable to be able to accomplish multiple diagnostic analyses such as enumerating nucleated blood cells and measuring hemoglobin concentration of a blood sample using the same reagent and concurrent measurements.
Among the many well known methods for hemoglobin determination, the cyanmethemoglobin method has been recommended as a standard by the International Committee for Standardization in Hematology. However, the presence of cyanide in the reagent waste has caused enormous environmental concern. In last ten years, a tremendous effort has been given to develop automated hemoglobin analysis methods without utilizing cyanide.
U.S. Pat. No. 5,242,832 (to Sakata) discloses a method using a cyanide-free lysing reagent for counting white blood cells and measuring the hemoglobin concentration in blood samples. PCT/US95/02897 (to Kim) discloses a cyanide-free method and reagent for determining hemoglobins in a whole blood sample. No capability of counting leukocytes, nor differentiating nucleated red blood cells is taught by Kim. U.S. Pat. No. 5,763,280 and 5,882,934 (to Li et al) disclose cyanide-free reagents for measuring hemoglobin in a blood sample, counting leukocytes, and differentiating leukocyte subpopulations. However, none of the above described hemoglobin measurement methods enables differentiation of nucleated red blood cells from other cell types.
Based on foregoing, there exists a need for a simple and less costly analysis method for differentiating and enumerating nucleated red blood cells. Furthermore, it is desirable to have a multifunctional test method for enumeration of nucleated blood cells, differentiation of nucleated red blood cells from other cell types, and measurement of hemoglobin concentration in one concurrent test.
In one embodiment, the present invention relates to a method of differentiating nucleated red blood cells from other cell types in a blood sample. The method comprises steps of mixing a blood sample with a lytic reagent to lyse red blood cells, and to form a blood sample mixture; measuring the blood sample mixture by a DC impedance measurement in a non-focused flow aperture, and obtaining a blood cell distribution of the blood sample mixture; differentiating nucleated red blood cells from other cell types; and reporting nucleated red blood cells in the blood sample. The non-focused flow aperture has an aperture aspect ratio of 0.7 and greater. Reporting nucleated red blood cells includes reporting the presence of nucleated red blood cells in the blood sample, and reporting numbers of nucleated red blood cells per one hundred of white blood cells in the blood sample, or numbers of nucleated red blood cells in an unit volume of the blood sample.
In a further embodiment, the present invention relates to a method of correcting white blood cell count. The method comprises the steps of mixing a blood sample with a lytic reagent to lyse red blood cells, and to form a blood sample mixture; measuring the blood sample mixture by a DC impedance measurement to obtain a blood cell distribution and a count of remaining blood cells; differentiating nucleated red blood cells and other interference materials from white blood cells; subtracting nucleated red blood cells and other interference materials from the count of remaining blood cells; and reporting a corrected white blood cell count in the blood sample. The method further comprises reporting nucleated red blood cells in the blood sample.
In another embodiment, the present invention relates to a method of concurrently differentiating nucleated red blood cells, enumerating white blood cells, and measuring hemoglobin concentration of a blood sample. The method comprises steps of mixing a blood sample with a lytic reagent to lyse red blood cells, and to form a blood sample mixture; measuring the blood sample mixture by a DC impedance measurement to obtain a blood cell distribution and a count of remaining blood cells; differentiating nucleated red blood cells and other interference materials from white blood cells; subtracting nucleated red blood cells and other interference materials from the count of remaining blood cells; measuring spectrophotometric absorbance of the blood sample mixture at a predetermined wavelength of a hemoglobin chromogen formed upon lysing the blood sample; reporting the nucleated red blood cells in the blood sample; reporting numbers of white blood cells in the blood sample; and reporting a hemoglobin concentration of the blood sample.